Most neoplasms, including bladder cancer, are thought to progress through a series of clinical histopathological stages. This progression is accompanied by specific genetic changes, which include activation of protooncogenes and loss of tumor suppressor genes. Recently, we defined another major event in bladder cancer progression outside of the nucleus: mitochondrial mutation. Studies in this proposal are aimed at the characterization of mitochondrial mutations in the development of bladder cancer and ultimately in developing new molecular detection strategies. First, a variety of lesions, including preinvasive and invasive tumors will be tested to identify the frequency and nature of mitochondrial mutations to develop a mitochondrial molecular progression model for bladder cancer. Second, functional studies will continue to identify the nature and function of specific mitochondrial mutations to better understand their role in the neoplastic process. Finally, we will continue development of assays that can detect mitochondrial mutations in urine DNA. Initial feasibility projects demonstrate that these studies will be greatly accelerated with the advent of high throughput mitochondrial mutation detection based on polymorphism ratio Sequencing (PRS). A combination of the above studies should provide important insights into the mitochondrial alterations associated with bladder tumor progression and their role in the neoplastic process. Additionally, characterization of highly susceptible mitochondrial regions will allow integration of critical targets for further development of molecular detection approaches.